How are knockout mice made?

Researchers begin by harvesting embryonic stem (ES) cells from early-stage mouse embryos four days after fertilization. ES cells are used because they are able to differentiate into nearly any type of adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse. In addition, ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested.

To produce knockout mice, researchers use one of two methods to insert artificial DNA into the chromosomes contained in the nuclei of ES cells. Both methods are carried out in vitro, that is in cultured cells grown in laboratory conditions.

In the first strategy, called gene targeting or homologous recombination researchers specifically manipulate a gene in the nucleus of an ES cell. Typically, this is done by introducing an artificial piece of DNA that shares identical, or homologous, sequence to the gene. This homologous sequence flanks the existing gene’s DNA sequence both upstream and downstream of the gene’s location on the chromosome. The cell’s own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA. Because the artificial DNA is inactive, bearing only a genetic tag, or “reporter gene,” designed for use in tracking, the swap eliminates, or “knocks out,” the function of the existing gene.


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